Next, we made a simulation in order to examine the size of the structure. The size of the tube must be small enough not to pass freely through anything. However, it must be large enough to pass through the desired product. The gate which made of DNA origami has negative electric charge. So if the gate is too small, target can't enter the Gate. According to simulation, our Gate size determined 24*24*33nm. This size is suitable to transport the target.

Cell-gate

Our project "Cell-gate" is left.
Our project is divided major three parts, Gate, Porter, and Membrane.
Gate is Cell-gate itself.
Porter is function to transport the target inside and outside cell membrane.
Membrane is liposome which is model of cell membrane.
We divided our project into above three group and did experiment.
On this page, we descript how we determined our robot design.

Design

Design of Gate

Size / Structure

The Gate has to connect inside and outside of the cell. So we decided to apply a hexagonal tube nanostructure made of DNA origami.

Next, we made a simulation in order to examine the size of the structure. The size of the tube must be small enough not to pass freely through anything. However, it must be large enough to pass through the desired product. The gate which made of DNA origami has negative electric charge. So if the gate is too small, target can't enter the Gate. According to simulation, our Gate size determined 24*24*33nm. This size is suitable to transport the target.

DNA origami

We used caDNAno to design the hexagonal tube structure.

Potential Barrier

What structure is most suitable to the Gate?
Gate have to connect inside to outside of the cell.
So Gate have to be the structure like a tube.
And it is desireble that is easy to make.
We decided to use the structure of the hexagonal tube.
The reason is because we think there is a reference on the hexagonal structure of DNA origami, for example "A logic-gated nanorobot for targeted transport of molecular payloads" (SM Douglas, I Bachelet, GM Church - Science Signalling, 2012), and can take advantage of that knowledge.

Design of Porter

Principle

When we consider making Cell Gate, there are two problems. One is how to pull the target oligonucleotide into Gate, and the other is how to pass the target. To solve these problems, we propose a nano structure of ssDNA and named it “Porter”. Porter stands in line in Gate, pulls the target, and transports it.

The Gate simulation shows that things by itself don’t come in Gate. So Porter has to pull and carry the target inside Gate.

We designed Porter having some loop structures when it hybridizes with the target. Porter has some complementary sequences to the target here and there. So after the target attaches to Porter at the end of Porter, Porter shrinks and pulls the target. Finally the target comes in Gate, otherwise unable to go through.

The inner Porter has more complementary sequences to the target and has higher bonding energy from the entrance of Gate. This design enables the target to move to the inner Porter because of the combination stability. In experiment, we apply the sequences below.

<DNA sequences>(実際の配列をお願いします) These are the sequences for electrophoresis, and only a part of the actual Porter sequence. These are the extracted region binding to the target. When planted in the gate, Porter has more spacer sequences of 10base at the root in addition to the sequences above, to reduce the Coulomb's force produced by the wall of Gate.

Simulation

We compared the ability to catch the target of Porter with that of toehold structure.

How to implement

Cell model

Cholesterol

We also have the aim of this GATE stab in the cell membrane, it can not sting in the cell membrane of normal hexagonal tube. However, We can create a tube with a different structure by exchanging some staple. We have designed a simple tube first,and I have to be attached anywhere in the structure to replace the staple. We can later add functionality to an existing structure using this method. We thought that to have an affinity for lipid membrane with the DNA that can be modified cholesterol on the side of the tube by this method. In addition, we have also designed DNA-like beard at the entrance of the tube. We expect the effect of electrostatically repel force with DNA which are not intended. Our tube is small,so we designed the tube to connect to each other and be long in order to easily confirmed using AFM. By replacing the staple, this structure is also removably.(その他の筒の絵)

Porter

What’s “Porter”?

When we consider making Cell Gate, there are two problems. One is how to pull the target oligonucleotide into Cylinder, and the other is how to pass the target. To solve these problems, we propose a nano structure of ssDNA and named it “Porter”. Porter stands in line in Cylinder, pulls the target, and transports it.

Loop structure

We did simulation to find the best length and place of Porter. The simulation shows that the target, which is negatively charged, can approach to Cylinder, only when there’s a certain distance between them. To enable the target to go enough close to Cylinder, Porter has to be longer than the distance and carry the target inside Cylinder. So we designed Porter having some loop structures when it hybridizes with the target. Porter has some complementary sequences to the target here and there. So after the target attaches to Porter at the end of Porter, Porter shrinks and pulls the target. Finally the target passes the distance otherwise unable to go through.

Transportation of the target

The inner Porter has more complementary sequences to the target and has higher bonding energy from the entrance of Cylinder. This design enables the target to move to the inner Porter because of the combination stability.

Experiment

In experiment, we apply the sequences below. We compared the effectiveness and efficiency of Porter with that of toehold structure. <DNA sequences>(実際の配列をお願いします) These are the sequences for electrophoresis, and only a part of the actual Porter sequence. These are the extracted region binding to the target. When planted in the gate, Porter has more spacer sequences of 10base at the root in addition to the sequences above, to reduce the Coulomb's force produced by the wall of Cylinder.

Membrane

Insert the mini-gate

We use liposomes as a model of cell membrane. First, we designed the composition of liposome and made the liposomes. And then, we prepared a preliminary step to insert cell gate into the liposome. We designed a smaller tube named “mini-gate” as a model of cell-gate. We attempted to insert it into our liposomes and we observed how “mini-gate” inserted into liposomes.

Modification of mini-gate

Similar to the actual cell gate, we also stretched single-stranded DNA of 10 bases that can be modified cholesterol from the side of “mini-gate” as Gate team did. We attached cholesterol to the side of the “mini-gate” using that single-stranded DNA, and expected that these enter into the hydrophobic portion of the liposome. Then, it is likely to that the tube stick to the liposome.

Penetrate with standing

However, we modified cholesterol to stick to the liposome, it is considered that the “mini- gate” would be in a state of lying on the liposome. Therefore, when we designed the mini- gate, we made one side too much for the extra M13 deliberately. We expect that the electrostatic symmetry of the tube is broken by this extra M13, and the tube penetrate the membrane standing vertically by repulsion.

( Inside the gate, a cascade of three single stranded DNAs is planted. We named the DNAs Selector1, Selector2, and Selector3 from the outside of the gate. In addition, another Selector, which is called Selector4, is in the liposome.
[In the gate] Selector1and 2 have complementary sequences to a target oligonucleotide here and there and consecutive adenine sequences in other portion. We made an attempt to capture a target distant from the gate with high specificity. So we lay out a long Selector1. By catching a target and making loops, it can shrink and go in the gate.

Selector3 is complementary to the target, but it is shorter than the target. When it binds to the target, the upper end of the target makes a toehold.
We designed the inner Selector has higher bonding energy. So once the outer-most ssDNA binds to a target, the target is passed to the inner-ones one by one.

[In liposome] Selector4 is perfectly complementary to the target. After the target reaches Selector3, Selector4 conveys the target into liposome.)

Name

Sequence(5' to 3')

Tm(°C)

target

* - ACTAGTGAGTGCAGCAGTCGTACCA

Selector1

AAAAAAAAAAAAAAAAATGGTACAAAAAAAAGACTGAAAAAAAACTGCA

30.6

Selector2

AAAAAAAAAAATGGTACAAAAGCTGCA

36.5

Selector3

TGGTACGACTGCTGCA

62.3

Selector4

TGGTACGACTGCTGCACTCA

68.0

Red-orange and blue-green regions are complementary DNA sequences.

Black region is added to differ the molecular weight of each sample(for distinguishing them during electrophoresis).

Tube

まず我々は円形断面のTubeを想定した。しかし、円形断面は力学的に見て強度が低い。そのためか、円形断面に関するDNAオリガミ構造体のデータも少なく、円形断面のTubeを作ることは難しそうであった。力学的に最も強度の高い構造は、三角形断面であるが、三角形断面は作成に必要な材料の数が多くなるなどの問題が挙げられる。また、三角形というある種特異的な断面を使用すると、目標物質の形状に関する汎用性が低下するおそれがある。断面はなるべく円に近いほうが良い。</br>
そこで考えたのが六角形断面、ハニカム構造の断面を持つ六角柱型のTubeである。六角形は三角形を6つ並べた構造をしているため、比較的強度が高く、建築物の構造にも使われ、自然界では蜂の巣の中などにその形を見出すことが出来る。また、六角柱型のDNAオリガミ構造体に関する参考文献（ショーンの論文を紹介）もあり、その知見を利用することが出来る。よってTubeは六角柱で構成することにする。</br>
ただの六角柱では細胞膜に刺さることはできない。そこで、筒の側面にコレステロールを修飾したDNAを生やし脂質膜と親和性を持たせる。さらにそのままでは、脂質の上に寝た状態でいることが安定になるといけないので、六角柱のDNAオリガミを作製する際、わざと片側にM13を余らせることにした。このことで対称性が崩れ、反発力によって、Tubeが立ったまま細胞膜を貫通することができる。
Gate should be able to transport the target with selector inside gate and go through cell membrane.
To transport the target with selector, we decided to make hexagonal tube as gate.
The reasons we adopted hexagonal tube as gate are that surfaces of hexagonal tube are suitable for being attached selector,
high strength of honeycomb structure are easy to be observed. To go through cell membrane, we placed the staple attached lipid on center of gate.</br>
We expect gate is introduced liposome simultaneously with creation of liposome.
In addition, we attached edge of the gate to adenine staple like a "mustache" to make easy watching by AFM and interrupt other DNA approaching.
We think because of our selector 1 is enough long, only target is transported into the gate. </br>
In addition to this, we made the cholesterol hexagonal tube. The reason that designed this tube is coupling into a liposome film using a characteristic of the cholesterol like a lipid. (cf.Figure 2.3 )
</br>

Membrane

We use liposomes as a model of cell membrane. To insert cell-gate into the liposome, we stretched out ssDNA of 10 nt from the side of the hexagonal tube. Then, we extend the complementary ssDNA, and modified the cholesterol at the end of them. We choose the cholesterol because cholesterol is strongly hydrophobic. We expect that cholesterol penetrate into the hydrophobic portion of the liposome. We confirmed the tube modifying the cholesterol by electrophoresis. We use fluorescein to confirm that the tube insert into the liposome correctly.
（細胞膜のモデルとして、リポソームを使用する。リポソームにセルゲートを刺さるようにするために、六角形筒の横から10塩基のシングルストランドDNAを伸ばした。さらに、相補的なシングルストランドDNAを伸ばして、それらの末端にコレステロール修飾を行った。コレステロールを修飾したのは、コレステロールが強い疎水性であるからである。コレステロールがリポソームの疎水性部分に入り込むことで、筒がリポソームに刺さりやすくなると考えた。コレステロール修飾は電気泳動で確かめた。筒がリポソームに刺さっているかを確かめる方法として、蛍光分子を利用する。）</br>
<a href=" http://openwetware.org/wiki/Biomod/2012/TeamSendaiA/Results_%26_Discussion#Membrane">Experiment page</a>